This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As a ubiquitous lysosomal pathway essential for degrading and recycling proteins and organelles, autophagy has been implicated in a broad spectrum of human disease including cancer, infection, liver disease, myopathy and neurodegeneration. However, the molecular machinery comprising the mammalian autophagy pathway and the molecular mechanism of how this machinery functions are largely unknown. Our long-term goal is to understand the molecular mechanism of the mammalian autophagy pathway and its relevance to human disease, for discovering much needed early diagnostic biomarkers and therapeutic targets. To achieve this goal, our overall objective is to first identify novel protein-protein interactions in the mammalian autophagy pathway and subsequently to determine the functions of these novel interactors. Beclin 1, the first reported mammalian autophagy protein, plays an important positive role in autophagy regulation and has been implicated in a variety of human physiology and pathology, including tumor suppression, neurodegeneration, development and aging. Therefore, for this study, we will investigate Beclin 1-mediated autophagy regulation so as to begin deciphering the molecular mechanism of mammalian autophagy. Guided by strong preliminary data, three specific aims will be pursued using a combination of a novel integrated mouse genetic-proteomic approach as well as biochemical and cell biological studies. In Aim 1, we will establish the framework of a context-dependent mammalian autophagy interactome through identifying Beclin 1-interacting proteins from living animals;in Aim 2, we will elucidate the molecular details of autophagy regulation by two novel Beclin 1-interacting proteins, Atg14L and Rubicon;and in Aim 3, we will explore the molecular details of autophagy regulation by a third novel Beclin 1-interactor and its cancer relevance. By establishing the framework of the mammalian autophagy interactome and unraveling the molecular details of autophagy regulation by Beclin 1 and its interacting proteins, this proposed work will not only provide insight into the role of autophagy in mammals but also generate novel potential targets for preventive and therapeutic interventions that may ultimately aid patients suffering from autophagy-related human disease.